The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present disclosure.
The seven serologically distinct botulinum neurotoxins produced by Clostridium botulinum, Clostridium argentiensis, and Clostridium baratti, (BoNT/A, /B, /C, /D, /E, /F, /G) are some of the most potent toxins known to mankind. These homologous toxins specifically target neurons and act through the interruption of neurotransmission. This interruption results in muscle paralysis, which in severe cases of intoxication leads to death from asphyxiation in humans and animals. Human botulism is typically caused by serotypes A, B, E, and occasionally F. Serotypes C and D cause toxicity only in non-human animals.
A single molecule of each toxin possesses three functional domains: receptor-recognition, transport and catalytic. The catalytic domains are Zn2+ metalloproteases that recognize and selectively cleave proteins involved in targeting of presynaptic vesicles and their fusion with the neuronal plasma membrane, in this way neurotoxins block neurotransmitter release into the synaptic cleft. Although there is a certain degree of homology between different clostridial neurotoxins, their catalytic domains recognize different substrates: BoNT/B, /D, /F and /G cleave synaptobrevin 2; BoNT/A, /C and /E cleave synaptosomal-associated protein of 25 kDa (SNAP25); BoNT/C cleaves syntaxin.
Botulinum neurotoxins are synthesized as single polypeptides approximately 1500 amino acids in length (Mr˜150 kDa), and then cleaved into heavy and light chains (Mr˜100 kDa, Mr˜50 kDa), which are held together by a disulfide bond. The light chain corresponds to the catalytic domain while heavy chains carry the receptor-recognition and transport domains, and are responsible for transport of corresponding light chains into the cytosol of neuronal cells.
Currently, botulinum neurotoxins are viewed as potent biological warfare agents. At the same time, botulinum neurotoxins of serotypes A and B are extensively used in medicine as drugs for the treatment of strabismus, blepharospasms, migraines and many other neurological conditions. The toxins are also commonly used as cosmetic agents.
In recent years, several researchers have used a series of synthetic BoNT/A peptides to map epitopes recognized by antisera from several species, including humans, and peptide displays of BoNT/A to map epitopes recognized by monoclonal antibodies. Others have reported epitopes recognized by neutralizing anti-BoNT antibodies. Thus far, however, epitope mapping efforts have focused on the heavy chains of botulinum neurotoxins.